Dataset Information

DNA sequence-based subtyping and evolutionary analysis of selected Salmonella enterica serotypes.

ABSTRACT: While serotyping and phage typing have been used widely to characterize Salmonella isolates, sensitive subtyping methods that allow for evolutionary analyses are essential for examining Salmonella transmission, ecology, and evolution. A set of 25 Salmonella enterica isolates, representing five clinically relevant serotypes (serotypes Agona, Heidelberg, Schwarzengrund, Typhimurium, and Typhimurium var. Copenhagen) was initially used to develop a multilocus sequence typing (MLST) scheme for Salmonella targeting seven housekeeping and virulence genes (panB, fimA, aceK, mdh, icdA, manB, and spaN). A total of eight MLST types were found among the 25 isolates sequenced. A good correlation between MLST types and Salmonella serotypes was observed; only one serotype Typhimurium var. Copenhagen isolate displayed an MLST type otherwise typical for serotype Typhimurium isolates. Since manB, fimA, and mdh allowed for the highest subtype discrimination among the initial 25 isolates, we chose these three genes to perform DNA sequencing of an additional 41 Salmonella isolates representing a larger diversity of serotypes. This "three-gene sequence typing scheme" allowed discrimination of 25 sequence types (STs) among a total of 66 isolates; STs correlated well with serotypes and allowed within-serotype differentiation for 9 of the 12 serotypes characterized. Phylogenetic analyses showed that serotypes Kentucky and Newport could each be separated into two distinct, statistically well supported evolutionary lineages. Our results show that a three-gene sequence typing scheme allows for accurate serotype prediction and for limited subtype discrimination among clinically relevant serotypes of Salmonella. Three-gene sequence typing also supports the notion that Salmonella serotypes represent both monophyletic and polyphyletic lineages.

SUBMITTER: Sukhnanand S

PROVIDER: S-EPMC1233967 | biostudies-literature | 2005 Aug

REPOSITORIES: biostudies-literature

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DNA sequence-based subtyping and evolutionary analysis of selected Salmonella enterica serotypes.

Sukhnanand Sharinne S Alcaine Sam S Warnick Lorin D LD Su Wan-Lin WL Hof Jessica J Craver Mary Pat J MP McDonough Patrick P Boor Kathryn J KJ Wiedmann Martin M

Journal of clinical microbiology 20050801 8

While serotyping and phage typing have been used widely to characterize Salmonella isolates, sensitive subtyping methods that allow for evolutionary analyses are essential for examining Salmonella transmission, ecology, and evolution. A set of 25 Salmonella enterica isolates, representing five clinically relevant serotypes (serotypes Agona, Heidelberg, Schwarzengrund, Typhimurium, and Typhimurium var. Copenhagen) was initially used to develop a multilocus sequence typing (MLST) scheme for Salmon ...[more]

PMID: 16081897

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Project description:BackgroundThe bacterium Salmonella enterica includes a diversity of serotypes that cause disease in humans and different animal species. Some Salmonella serotypes show a broad host range, some are host restricted and exclusively associated with one particular host, and some are associated with one particular host species, but able to cause disease in other host species and are thus considered "host adapted". Five Salmonella genome sequences, representing a broad host range serotype (Typhimurium), two host restricted serotypes (Typhi [two genomes] and Paratyphi) and one host adapted serotype (Choleraesuis) were used to identify core genome genes that show evidence for recombination and positive selection.ResultsOverall, 3323 orthologous genes were identified in all 5 Salmonella genomes analyzed. Use of four different methods to assess homologous recombination identified 270 genes that showed evidence for recombination with at least one of these methods (false discovery rate [FDR] <10%). After exclusion of genes with evidence for recombination, site and branch specific models identified 41 genes as showing evidence for positive selection (FDR <20%), including a number of genes with confirmed or likely roles in virulence and ompC, a gene encoding an outer membrane protein, which has also been found to be under positive selection in other bacteria. A total of 8, 16, 7, and 5 genes showed evidence for positive selection in Choleraesuis, Typhi, Typhimurium, and Paratyphi branch analyses, respectively. Sequencing and evolutionary analyses of four genes in an additional 42 isolates representing 23 serotypes confirmed branch specific positive selection and recombination patterns.ConclusionOur data show that, among the four serotypes analyzed, (i) less than 10% of Salmonella genes in the core genome show evidence for homologous recombination, (ii) a number of Salmonella genes are under positive selection, including genes that appear to contribute to virulence, and (iii) branch specific positive selection contributes to the evolution of host restricted Salmonella serotypes.

Project description:ABSTRACT Six subspecies are currently recognized in Salmonella enterica. Subspecies I (subspecies enterica) is responsible for nearly all infections in humans and warm-blooded animals, while five other subspecies are isolated principally from cold-blooded animals. We sequenced 21 phylogenetically diverse strains, including two representatives from each of the previously unsequenced five subspecies and 11 diverse new strains from S. enterica subspecies enterica, to put this species into an evolutionary perspective. The phylogeny of the subspecies was partly obscured by abundant recombination events between lineages and a relatively short period of time within which subspeciation took place. Nevertheless, a variety of different tree-building methods gave congruent evolutionary tree topologies for subspeciation. A total of 285 gene families were identified that were recruited into subspecies enterica, and most of these are of unknown function. At least 2,807 gene families were identified in one or more of the other subspecies that are not found in subspecies I or Salmonella bongori. Among these gene families were 13 new candidate effectors and 7 new candidate fimbrial clusters. A third complete type III secretion system not present in subspecies enterica (I) isolates was found in both strains of subspecies salamae (II). Some gene families had complex taxonomies, such as the type VI secretion systems, which were recruited from four different lineages in five of six subspecies. Analysis of nonsynonymous-to-synonymous substitution rates indicated that the more-recently acquired regions in S. enterica are undergoing faster fixation rates than the rest of the genome. Recently acquired AT-rich regions, which often encode virulence functions, are under ongoing selection to maintain their high AT content. IMPORTANCE We have sequenced 21 new genomes which encompass the phylogenetic diversity of Salmonella, including strains of the previously unsequenced subspecies arizonae, diarizonae, houtenae, salamae, and indica as well as new diverse strains of subspecies enterica. We have deduced possible evolutionary paths traversed by this very important zoonotic pathogen and identified novel putative virulence factors that are not found in subspecies I. Gene families gained at the time of the evolution of subspecies enterica are of particular interest because they include mechanisms by which this subspecies adapted to warm-blooded hosts.

Project description:Serotyping has traditionally been used for subtyping of non-typhoidal Salmonella (NTS) isolates. However, its discriminatory power is limited, which impairs its use for epidemiological investigations of source attribution. Whole-genome sequencing (WGS) analysis allows more accurate subtyping of strains. However, because of the relative newness and cost of routine WGS, large-scale studies involving NTS WGS are still rare. We aimed to revisit the big picture of subtyping NTS with a public health impact by using traditional serotyping (i.e. reaction between antisera and surface antigens) and comparing the results with those obtained using WGS. For this purpose, we analysed 18 282 sequences of isolates belonging to 37 serotypes with a public health impact that were recovered in the USA between 2006 and 2017 from multiple sources, and were available at the National Center for Biotechnology Information (NCBI). Phylogenetic trees were reconstructed for each serotype using the core genome for the identification of genetic subpopulations. We demonstrated that WGS-based subtyping allows better identification of sources potentially linked with human infection and emerging subpopulations, along with providing information on the risk of dissemination of plasmids and acquired antimicrobial resistance genes (AARGs). In addition, by reconstructing a phylogenetic tree with representative isolates from all serotypes (n=370), we demonstrated genetic variability within and between serotypes, which formed monophyletic, polyphyletic and paraphyletic clades. Moreover, we found (in the entire data set) an increased detection rate for AARGs linked to key antimicrobials (such as quinolones and extended-spectrum cephalosporins) over time. The outputs of this large-scale analysis reveal new insights into the genetic diversity within and between serotypes; the polyphyly and paraphyly of certain serotypes may suggest that the subtyping of NTS to serotypes may not be sufficient. Moreover, the results and the methods presented here, leading to differentiation between genetic subpopulations based on their potential risk to public health, as well as narrowing down the possible sources of these infections, may be used as a baseline for subtyping of future NTS infections and help efforts to mitigate and prevent infections in the USA and globally.

Project description:Metagenomics analysis of food samples promises isolation-independent detection and subtyping of foodborne bacterial pathogens in a single workflow. The selective concentration of Salmonella genomic DNA by immunomagnetic separation (IMS) and multiple displacement amplification (MDA) shortened the time for culture enrichment of Salmonella-spiked raw chicken breast samples by over 12 h while permitting serotyping and high-fidelity single nucleotide polymorphism (SNP) typing of the pathogen using short shotgun sequencing reads. The herein-termed quasimetagenomics approach was evaluated on Salmonella-spiked lettuce and black peppercorn samples as well as retail chicken parts naturally contaminated with different serotypes of Salmonella Culture enrichment of between 8 and 24 h was required for detecting and subtyping naturally occurring Salmonella from unspiked chicken parts compared with 4- to 12-h culture enrichment when Salmonella-spiked food samples were analyzed, indicating the likely need for longer culture enrichment to revive low levels of stressed or injured Salmonella cells in food. A further acceleration of the workflow was achieved by real-time nanopore sequencing. After 1.5 h of analysis on a potable sequencer, sufficient data were generated from sequencing the IMS-MDA products of a cultured-enriched lettuce sample to enable serotyping and robust phylogenetic placement of the inoculated isolate.IMPORTANCE Both culture enrichment and next-generation sequencing remain time-consuming processes for food testing, whereas rapid methods for pathogen detection are widely available. Our study demonstrated a substantial acceleration of these processes by the use of immunomagnetic separation (IMS) with multiple displacement amplification (MDA) and real-time nanopore sequencing. In one example, the combined use of the two methods delivered a less than 24-h turnaround time from the collection of a Salmonella-contaminated lettuce sample to the phylogenetic identification of the pathogen. An improved efficiency such as this is important for further expanding the use of whole-genome and metagenomics sequencing in the microbial analysis of food. Our results suggest the potential of the quasimetagenomics approach in areas where rapid detection and subtyping of foodborne pathogens are important, such as for foodborne outbreak response and the precision tracking and monitoring of foodborne pathogens in production environments and supply chains.

Dataset Information

DNA sequence-based subtyping and evolutionary analysis of selected Salmonella enterica serotypes.

Publications

DNA sequence-based subtyping and evolutionary analysis of selected Salmonella enterica serotypes.

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